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Uptake of Irradiated Tumor Cells by Dendritic Cells
No full textSlide 5-35: Uptake of irradiated tumor cells by dendritic cells primarily takes place following intravenous injectionhttps://digitalcommons.rockefeller.edu/nkt-cells/1033/thumbnail.jp
Diverse Kappa Opioid Receptor Agonists: Relationships Between Signaling and Behavior
Get PDFThe opioid system, comprised mainly of the three opioid receptors (kappa, mu and delta) and their endogenous neuropeptide ligands (dynorphin, endorphin and enkephalin, respectively), mediates mood and reward. Activation of the mu opioid receptor is associated with positive reward and euphoria, while activation of the kappa opioid receptor (KOR) has the opposite effect. Activation of the KOR causes a decrease in dopamine levels in reward-related regions of the brain, and can block the rewarding effects of various drugs of abuse, making it a potential drug target for addictive diseases. KOR agonists are of particular interest for the treatment of cocaine and other psychostimulant addictions, because there are currently no available medications for these diseases. Studies in humans and animals, however, have shown that activation of the KOR also causes negative side effects such as hallucinations, aversion and sedation. Several strategies are currently being employed to develop KOR agonists that block the rewarding effects of drugs of abuse with fewer side effects, including KOR agonists with unique pharmacology. The goal of the research presented here was to profile the signaling pathways activated by KOR agonists and to investigate relationships between unique pharmacology and animal models of KOR-mediated behaviors, in order to better understand how to target the KOR for therapeutic use
Testing the T-loop Model of Telomeric End Protection
Get PDFTelomeres are the key structures that protect the ends of linear chromosomes. Although they are often thought about in the context of cellular aging, their most important role is actually to protect the end of the DNA from being mis-interpreted as a site of DNA damage. Telomeres are thought to accomplish this through the action of shelterin. Shelterin is a multi-protein complex where each subunit is dedicated to a specific role in repressing a form of DNA damage signaling, repair, or recruiting telomerase to extend the telomere end. One of the critical anchor points of shelterin is a protein known as TRF2. TRF2 is necessary to protect telomeres from becoming fused by non-homologous end-joining, and from one of the two main DNA damage signaling pathways, in this case the one driven by ATM and CHK2. It is thought to do this by rearranging the very 3\u27 end of the DNA into a duplex loop – known as the t-loop. Together, TRF2 and t-loops are the main pillars of the t-loop model of end-protection, which is the focus of this thesis. The first part of this thesis presents an overview of telomere protection and focuses specifically on what is known about end-protection in mammalian cells. From there, we test an alternative model of telomere end-protection, and find it to be unsubstantiated. We next analyze how TRF2 contributes to t-loop formation, including whether TRF2 cooperates with other shelterin components, uses non-shelterin factors, and which domains of TRF2 contribute. Finally, we try to understand how t-loops are made, and whether there are any external factors that assist TRF2, or whether TRF2 is self-sufficient in repressing signaling, fusions, and forming t-loops. We then discuss the evolution of telomeres which serves as an important reference point towards understanding the greater context of the t-loop model, and its plausibility. The appendix discusses attempts to push the resolution of t-loop imaging in the context of whole cells. The work presented here is of relevance to understanding the central mechanism of telomere end protection. What t-loops do – if anything – and how they are made is a question that is at the heart of telomere biology
Time-Restricted Feeding Extends Longevity in Drosophila Melanogaster
Get PDFTime-restricted feeding (TRF) is a dietary intervention in which daily feeding patterns align with behavioral patterns, synchronizing feeding times with periods of higher activity, e.g. humans eating only during the day or rodents (nocturnal animals) at night. TRF has been shown to improve cardiac health in Drosophila melanogaster, reduce metabolic markers in rodent models, and reduce glycemic indices in prediabetic men. However, the mechanism and long-term effects of this intervention remain elusive. To understand the effect of TRF on longevity we used the fruit fly Drosophila melanogaster, which is a useful model for longevity, sleep and circadian studies because of its wellestablished sleep behavior, tractable genetics, and short lifespan. We found that TRF extends longevity of fruit flies only in mated females, while showing no effect on mated males or virgin females. We measured the amount of food consumed by flies on TRF and confirmed that TRF does not act through caloric restriction, which has been previously shown to extend longevity. Remarkably, animals undergoing TRF eat more, yet have lower body weight in comparison to animals on constant food. TRF-mediated lifespan extension is dependent on the molecular clock, as arrhythmic clock mutants fail to respond to TRF under light-dark conditions, suggesting that TRF may act as a zeitgeber to improve the animals\u27 health by coordinating activity patterns with food availability. In addition to its effect on wild-type animals, TRF also improves longevity in animals with reduced lifespan, such as sleep mutants. Further studies show that TRF changes the sleep architecture of wild-type females by increasing the amount of day sleep, while also promoting integrity of the blood-brain barrier. TRF life-extension effects show that this dietary intervention has potential to reveal a deeper understanding of the biology of ageing and how it interacts with feeding and circadian rhythms, placing a larger emphasis on time of intake rather than calories
Representations of Reward and Movement in Drosophila Dopaminergic Neurons
Get PDFThe neuromodulator dopamine is known to influence both immediate and future behavior, motivating and invigorating an animal\u27s ongoing movement but also serving as a reinforcement signal to instruct learning. Yet it remains unclear whether this dual role of dopamine involves the same dopaminergic pathways. Although reward-responsive dopaminergic neurons display movement-related activity, debate continues as to what features of an individual\u27s experience these motor-correlates correspond and how they influence concurrent behavior. The mushroom body, a prominent neuropil in the brain of the fruit fly Drosophila melanogaster, is richly innervated by dopaminergic neurons that play an essential role in the formation of olfactory associations. While dopaminergic neurons respond to reward and punishment to drive associative learning, they have also been implicated in a number of adaptive behaviors and their activity correlates with the behavioral state of an animal and its coarse motor actions. Here, we take advantage of the concise circuit architecture of the Drosophila mushroom body to investigate the nature of motor-related signals in dopaminergic neurons that drive associative learning. In vivo functional imaging during naturalistic tethered locomotion reveals that the activity of different subsets of mushroom body dopaminergic neurons reflects distinct aspects of movement. To gain insight into what facets of an animal\u27s experience are represented by these movement-related signals, we employed a closed loop virtual reality paradigm to monitor neural activity as animals track an olfactory stimulus and are actively engaged in a goal-directed and sensory-motivated behavior. We discover that odor responses in dopaminergic neurons correlate with the extent to which an animal tracks upwind towards the fictive odor source. In different experimental contexts where distinct motor actions were required to track the odor, dopaminergic neurons become emergently linked to the behavioral metric most relevant for effective olfactory navigation. Subsets of dopaminergic neurons were correlated with the strength of upwind tracking regardless of the identity of the odor and remained so even after the satiety state of an animal was altered. We proceed to demonstrate that transient inhibition of dopaminergic neurons that are positively correlated with upwind tracking significantly diminishes the normal approach responses to an appetitive olfactory cue. Accordingly, activation of those same dopaminergic neurons enhances approach to an odor and even drives upwind tracking in clean air alone. Together, these results reveal that the same dopaminergic pathways that convey reinforcements to instruct learning also carry representations of an animal\u27s moment-by-moment movements and actively influence behavior. The complex activity patterns of mushroom body dopaminergic neurons therefore represent neither purely sensory nor motor variables but rather reflect the goal or motivation underlying an animal\u27s movements. Our data suggest a fundamental coupling between reinforcement signals and motivation-related locomotor representations within dopaminergic circuitry, drawing a striking parallel between the mushroom body dopaminergic neurons described here and the emerging understanding of mammalian dopaminergic pathways. The apparent conservation in dopaminergic neuromodulatory networks between mammals and insects suggests a shared logic for how neural circuits assign meaning to both sensory stimuli and motor actions to generate flexible and adaptive behavior
Hilda Huang, Piano
No full textHilda Huang, piano, performed Beethoven @250. Performing Bach: Toccata in C Minor, BWV 911; Bartók: Suite, Op. 14; Beethoven: Piano Sonata No. 24, op. 78 in F-sharp Major (À Thérèse); Bach: Overture in the French style, BWV 831.
Image Credit Verena Brüninghttps://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1005/thumbnail.jp
Identification of Previously Unknown Interactions Between G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins
Get PDFG protein-coupled receptors (GPCRs) are known to interact with several other classes of integral membrane proteins. However, the extent of these interactions and their role in regulating GPCR-mediated transmembrane signaling is not well understood. For example, receptor activity-modifying proteins (RAMPs), a family of single transmembrane proteins with only three members, are ubiquitously expressed and have been shown to interact with several different GPCRs. Most research to date has focused on the ability of RAMPs to modulate the function of several GPCRs in the secretin-like GPCR family. GPCR-RAMP interactions were shown to affect the ligand binding affinity of two different secretin-like GPCRs, causing the functional diversity of the GPCRs to be driven by an interacting protein. Yet, potential direct interactions among the three known RAMPs and hundreds of non-olfactory GPCR has never been investigated and whether RAMP-GPCR interactions are widespread remains an open question. To determine the breadth of GPCR-RAMP interactions, we first investigated the global coexpression and coevolution between GPCRs and RAMPs. On the one hand, if many GPCRs interact with RAMPs we would except to see statistically significant coexpression and coevolution in comparison to random gene pairs. On the other hand, if GPCR-RAMP interactions are limited to a small number of genes, then averaged coexpression and coevolution would be similar to that expected by chance. To calculate coexpression, we analyzed an RNASeq database of human transcriptomes across 53 different tissues and found that coexpression between non-olfactory GPCRs and RAMPs is significantly higher than random gene pairs. We also analyzed genomic data from all currently available sequenced organisms to calculate the coevolution between non-olfactory GPCRs and RAMPs. We discovered that GPCRs and RAMPs have a significant percentage of shared species and significantly correlated phylogenetic trees. Our results support the hypothesis that GPCRs interact globally with RAMPs. Only a handful of GPCR-RAMP interactions have been reported to date, but our coexpression and coevolution analysis suggested that additional GPCRs interact with RAMPs. To begin to address the potential for direct interactions among the three known RAMPs and hundreds of GPCRs, we developed a highly multiplexed immunoassay using a suspension bead array (SBA) assay designed to detect RAMP-GPCR complexes. We engineered three epitopetagged RAMPs and 23 epitope-tagged GPCRs, including all members of the secretin-like family of GPCRs, as well as eight other GPCRs. We then used 64 antibodies raised against native RAMPs and GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to detect and measure all possible combinations of interaction among the 23 GPCRs and three RAMPs from detergent-solubilized lysates. We also used the epitope-tagged constructs to verify a collection of antibodies that target native GPCRs and RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. Using in situ proximity ligation assay, we verified a subset of these novel GPCR-RAMP interactions in cell membranes. The results of the SBA assay provide a complete interactome of secretin-like GPCRs with RAMPs. GPCR-RAMP interactions are more common than previously appreciated, and the SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues
Transmission of HIV-1 to T Blasts
No full textSlide 3-7: Transmission of HIV-1 to T blasts: comparison of DCs with DC-SING transfectantshttps://digitalcommons.rockefeller.edu/immunodeficiency-disease/1006/thumbnail.jp
Pathogenesis & Protection
No full textSlide 3-5: Pathogenesis and protectionhttps://digitalcommons.rockefeller.edu/immunodeficiency-disease/1004/thumbnail.jp
DEC-205 on Dendritic Cells in Lymph Node T Areas
No full textSlide 4-11: DEC-205 on dendritic cells in lymph node T areashttps://digitalcommons.rockefeller.edu/endocytosis/1010/thumbnail.jp